CN116986976B - Hazian diterpenoid compound, trichoderma hook fermentation liquor extract, pesticide, and preparation method and application thereof - Google Patents
Hazian diterpenoid compound, trichoderma hook fermentation liquor extract, pesticide, and preparation method and application thereof Download PDFInfo
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- CN116986976B CN116986976B CN202311251991.6A CN202311251991A CN116986976B CN 116986976 B CN116986976 B CN 116986976B CN 202311251991 A CN202311251991 A CN 202311251991A CN 116986976 B CN116986976 B CN 116986976B
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- trichoderma
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- 241000223259 Trichoderma Species 0.000 title claims abstract description 35
- 239000000284 extract Substances 0.000 title claims abstract description 26
- 238000000855 fermentation Methods 0.000 title claims abstract description 19
- 230000004151 fermentation Effects 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000000575 pesticide Substances 0.000 title claims abstract description 8
- -1 diterpenoid compound Chemical class 0.000 title abstract description 7
- 150000001875 compounds Chemical class 0.000 claims abstract description 45
- 241000233866 Fungi Species 0.000 claims abstract description 22
- 201000010099 disease Diseases 0.000 claims abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 12
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 9
- 208000035240 Disease Resistance Diseases 0.000 claims abstract description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 241000196324 Embryophyta Species 0.000 claims description 11
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 241000123650 Botrytis cinerea Species 0.000 claims description 4
- 235000016623 Fragaria vesca Nutrition 0.000 claims description 4
- 240000009088 Fragaria x ananassa Species 0.000 claims description 4
- 235000011363 Fragaria x ananassa Nutrition 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 4
- 230000006835 compression Effects 0.000 claims description 4
- 238000007906 compression Methods 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 4
- 239000003208 petroleum Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 241000193738 Bacillus anthracis Species 0.000 claims description 3
- 241000227728 Trichoderma hamatum Species 0.000 claims description 3
- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 2
- 244000105624 Arachis hypogaea Species 0.000 claims description 2
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 2
- 235000018262 Arachis monticola Nutrition 0.000 claims description 2
- 241000223218 Fusarium Species 0.000 claims description 2
- 241000233616 Phytophthora capsici Species 0.000 claims description 2
- 241000813090 Rhizoctonia solani Species 0.000 claims description 2
- 241000221696 Sclerotinia sclerotiorum Species 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 235000020232 peanut Nutrition 0.000 claims description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 7
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 238000012512 characterization method Methods 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 238000005481 NMR spectroscopy Methods 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 241000258957 Asteroidea Species 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 229930000044 secondary metabolite Natural products 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000012258 culturing Methods 0.000 description 4
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000235349 Ascomycota Species 0.000 description 3
- 241001092371 Bergenia Species 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002983 circular dichroism Methods 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- NMUSYJAQQFHJEW-UHFFFAOYSA-N 5-Azacytidine Natural products O=C1N=C(N)N=CN1C1C(O)C(O)C(CO)O1 NMUSYJAQQFHJEW-UHFFFAOYSA-N 0.000 description 2
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000662429 Fenerbahce Species 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 241000499912 Trichoderma reesei Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 229960002756 azacitidine Drugs 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 2
- 235000013923 monosodium glutamate Nutrition 0.000 description 2
- 239000004223 monosodium glutamate Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 241000221781 Hypocreaceae Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241001326533 Sordariomycetes Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000000443 biocontrol Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000000110 cooling liquid Substances 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 230000001863 plant nutrition Effects 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
- C07C49/703—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups
- C07C49/723—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups polycyclic
- C07C49/727—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups polycyclic a keto group being part of a condensed ring system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N45/00—Biocides, pest repellants or attractants, or plant growth regulators, containing compounds having three or more carbocyclic rings condensed among themselves, at least one ring not being a six-membered ring
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/80—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/56—Ring systems containing bridged rings
- C07C2603/86—Ring systems containing bridged rings containing four rings
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Environmental Sciences (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Dentistry (AREA)
- Biotechnology (AREA)
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to the technical field of pesticides, and particularly relates to a halkane diterpenoid compound, trichoderma hook fermentation broth extract, a pesticide, a preparation method and application thereof. The invention provides Ha Ciwan diterpenoid compounds extracted and separated from trichoderma hook and an extract containing the compounds, and the compounds and the extract have good inhibition effect on various agricultural disease fungi. Thus, they are useful for inhibiting agricultural disease fungi and improving plant disease resistance. The technical scheme of the invention is beneficial to the popularization and the efficient utilization of trichoderma hook and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of pesticides, and particularly relates to a halkane diterpenoid compound, trichoderma hook fermentation broth extract, a pesticide, a preparation method and application thereof.
Background
Trichoderma genusTrichoderma spp.) Fungi are widely used for biological control of plant diseases, and the biological control mechanism is various. The antibiotic effect is one of the main biocontrol mechanisms of the trichoderma fungi, and researches show that the secondary metabolite produced by the trichoderma fungi not only can directly inhibit the growth of pathogenic bacteria, but also can trigger a plant defense system or promote the plant nutrition growth so as to improve the disease resistance of plants.
Trichoderma hookTrichoderma hamatum) Is ascomycetes (Ascomycota), ascomycetes (Sordariomycetes), hypocrea (Hypocreatles), hypocreaceae (Hypocreateae), trichodermaTrichodermasp.) fungi. Currently, there is a lack of comprehensive research on the secondary metabolites of trichoderma reesei. Thus, the active ingredients for biological control of plant diseases for trichoderma hook have not been determined. This is disadvantageous for the wide and efficient use of trichoderma reesei.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a halzane diterpenoid compound, a preparation method and application thereof, and aims to provide an active ingredient in a secondary metabolite of trichoderma hook and application thereof.
A compound of formula I, or a stereoisomer thereof, or a crystalline form thereof, or a salt thereof, or a derivative thereof:
formula I.
The invention also provides a trichoderma hook fermentation liquor extract, which comprises the compound.
Preferably, the trichoderma hook isTrichoderma hamatum。
The invention also provides application of the compound, or a stereoisomer, a crystal form, a salt or a derivative thereof, or the trichoderma hook fermentation broth extract for improving plant disease resistance.
The invention also provides application of the compound, or a stereoisomer, a crystal form, a salt or a derivative thereof, or the trichoderma hook fermentation broth extract for inhibiting agricultural disease fungi.
Preferably, the agricultural disease fungus comprises at least one of the following fungi: gibberella Tritici, phytophthora capsici, sclerotinia sclerotiorum, botrytis cinerea, rhizoctonia solani, corn stalk base rot, strawberry anthrax, and peanut brown spot.
The invention also provides a pesticide for improving plant disease resistance, which is prepared by taking the compound, or a stereoisomer, a crystal form, a salt or a derivative thereof, or the trichoderma hook fermentation liquor extract as an active ingredient.
The invention also provides a preparation method of the compound, which comprises the following steps:
step 1, extracting ethyl acetate part extract from fermentation broth of trichoderma hook;
step 2, coarsely separating the ethyl acetate part extract by column chromatography, wherein the volume ratio of the column chromatography is 30: 1-1: 1, petroleum ether-acetone is used as a mobile phase, 8 components are obtained through separation, and the components are sequentially recorded as Fr.1-Fr.8;
step 3, fr.3 is eluted by a radial compression column, and eluent is methanol aqueous solution with the volume fraction of 50-100% to obtain 6 components, which are sequentially recorded as Fr.3-1-Fr.3-6;
step 4, eluting Fr.3-4 by a gel column, wherein the eluent is methanol to obtain 5 components, which are sequentially recorded as Fr.3-4-1 to Fr.3-4-5;
and 5, taking Fr.3-4-3 for purification to obtain the final product.
Preferably, the trichoderma hook isTrichoderma hamatum。
Preferably, the preparation method of the ethyl acetate part extract comprises the following steps: separating fermentation broth of Trichoderma hook, soaking in ethyl acetate, extracting with ethyl acetate, mixing ethyl acetate phases, and concentrating under reduced pressure.
The invention separates a new compound (compound shown in formula I) from the trichoderma hook secondary metabolite, and determines the absolute configuration of the compound through the comprehensive analysis of spectrum data such as Nuclear Magnetic Resonance (NMR), electrospray mass spectrum (ESIMS), circular Dichroism (CD) and the like. The compound has remarkable inhibitory activity on fungi such as sclerotium of rape and botrytis cinerea of cucumber, and has good inhibitory activity on fungi such as corn stalk rot and strawberry anthracnose. Thus, the compound or an extract containing the compound can be used for improving disease resistance of plants. The technical scheme of the invention is beneficial to the popularization and the efficient utilization of trichoderma hook and has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a representation of ESI-MS characterization of a compound of formula I;
FIG. 2 is a graph showing the results of UV spectral characterization of compounds of formula I;
FIG. 3 is a graph showing the IR spectrum characterization of the compound of formula I;
FIG. 4 is a compound of formula I 1 H NMR characterization results;
FIG. 5 is a compound of formula I 13 C NMR characterization results;
FIG. 6 is a compound of formula I 1 H- 1 H COSY characterization results;
FIG. 7 shows the HSQC spectrum characterization of the compounds of formula I;
FIG. 8 is a graph showing the results of HMBC spectra of a compound of formula I;
FIG. 9 is a NOESY spectrum characterization of a compound of formula I;
fig. 10 shows the results of the calculation and experimental characterization of circular dichroism.
Detailed Description
In the following examples and experimental examples, reagents and materials not specifically described are commercially available, and specific ones are as follows:
1. reagent(s)
Fungus No. 2 medium: maltose 20 g; monosodium glutamate 10 g; glucose 10 g; yeast extract 3 g; corn steep liquor 1 g; mannitol 20 g; KH (KH) 2 PO 4 0.5 g；MgSO 4 . 7H 2 O 0.3 g；CaCO 3 20 g; 5-azacytidine 10 mg; distilled water 1L;
deuterated methanol (adamas reagent limited); deuterated chloroform (adamas beta (Shanghai) chemical company, inc.); analytical grade methanol (Shanghai Starfish high purity solvent Co., ltd.); petroleum ether (Shanghai Starfish high purity solvent Co., ltd.); acetone (colone chemicals limited, adult city); preparation grade acetonitrile (Shanghai Starfish high purity solvent Co., ltd.); dichloromethane (Shanghai Starfish high purity solvent Co., ltd.); preparation grade methanol (starfish high purity solvent limited); analytical grade ethyl acetate (starfish high purity solvent Co., ltd.).
2. Instrument for measuring and controlling the intensity of light
Column chromatography silica gel (200-300 mesh) (Qingdao ocean chemical plant); diaion HP-20 (Mitsubishi chemical corporation, japan); MCI filler (mitsubishi chemical company, japan); a water-proof electrothermal constant temperature incubator (Shanghai medical instruments Co., ltd.); ultrapure water machine (Sichuan Upoaching technology Co., ltd.); QExactive UHMR composite quadrupole-orbitrap mass spectrometer (Simer Feier Co., USA); silica gel (200-300 mesh, qingdao sea wave silica gel desiccant Co., ltd.); LDZX-50KBS vertical pressure steam sterilizer (Shanghai Shen An medical instruments factory); axial dynamic compression column (China Jiangsu Hanbang technology); NU3000 type ultraviolet detector (china Jiangsu hanbang technology); vacuum drying oven (DZF type, beijing Uygur instrument Co., ltd.); kromasil100-5-C18, (10X 250 mm, 4.5 μm, akzo Nobel, sweden); rapidly preparing a liquid phase apparatus (Santai technology instruments Co., ltd.); bruker Bruker-Assnd-700-MHz nuclear magnetic resonance apparatus (Bruker, germany); high speed centrifuges (Hunan Instrument centrifuges, inc.); NP7000 semi-preparative high performance liquid chromatograph (Jiangsu hanbang technology, china); bruker Bruker-Assend-600-MHz ultra-pure water (Sichuan UpUpUpULTRACTICH Co., ltd.) nuclear magnetic resonance apparatus (Bruker, germany); vacuum drying oven (DZF type, beijing Uygur instrument Co., ltd.); perkin-Elmer 241MC polarimeter (Perkin Elmer, inc.); chirascan round two chromatograph (applied photophysics limited, uk); cary 600 FT-IR infrared spectrometer (agilent technologies, california, usa); SL 302N-type ten-thousandth electronic balance (Shanghai Minqiao precision scientific instruments limited); SHZ-D (III) circulating water type vacuum pump (incorporated by Hua instrument Limited); DLSB-5L/20 type cryogenic cooling liquid circulating pump (incorporated by reference, instrument Co., ltd.).
EXAMPLE 1 isolation and identification of Compounds of formula I
1. Isolation of the Compounds of formula I
1. Experimental strains
Trichoderma hookTrichoderma hamatum) GenBank accession number (OR 553890).
In this example, trichoderma hook was isolated as follows:
the experimental strain is obtained by separating fresh plants of bergenia (bergenia tenacissima) which is a traditional Chinese medicine, fresh leaves and rhizomes of the bergenia tenacissima are taken and washed clean in flowing water, and moisture is dried. In a sterile operation room, the rootstock and the leaf are respectively soaked in 75% alcohol for 1min in culture dishes, and then are rinsed with sterile water for 3 times in 3 culture dishes by forceps, and then are taken out and fully dried by sterile filter paper. A blank control group is set, 200 microliters of sterile water of the last cleaning sample is taken by a liquid-transferring gun, the sterile water is uniformly smeared on a PDA (potato 200g, glucose 20g, agar 20g, distilled water 1L) flat plate by a metal coating rod to serve as a control, and the flat plate is inversely cultured in a constant-temperature incubator at 28 ℃. If no colony grows on the culture dish of the control group, the surface is thoroughly disinfected. The test group uses a sterile filter paper sheet to suck the surface moisture of the sample, then uses a sterile scalpel to cut the sample into small pieces, cuts the leaves into square blocks with the length of 5mm multiplied by 5mm, cuts the rhizome into fragments with the length of 0.5cm, and cuts the rhizome from the middle. The tissue blocks are inoculated into PDA culture medium respectively, each dish is 2 blocks, the incision is tightly attached to the PDA culture medium, the PDA culture medium is sealed by a sealing film, and the PDA culture medium is cultured in a constant temperature incubator at 28 ℃.
And (3) observing the culture dish every day, marking the culture dish after bacterial colony grows out, inoculating hypha into a new culture medium from a sample incision by using an inoculating loop, culturing and observing, primarily judging whether the culture dish is a single strain or not by using the color and the shape of the hypha, and if not, continuously picking and purifying the hypha respectively until each strain reaches a separation culture condition. And after separating the strains, continuously culturing the strains for 3 to 5 days in a 28 ℃ incubator, and preserving the strains in a refrigerator at 4 ℃. The fungus b-3 is identified by the division of biological engineering (Shanghai) and completed (GenBank accession number OR553890, serial number NR 134371.1) and is identified as Trichoderma hamatum by BLAST comparison. Is stored in the biochemical pharmacy laboratory of the university of Chinese medicine and college of pharmacy.
2. Expansion culture, extraction and separation of strain
Inoculating the experimental strain to PDA culture medium, activating for 3 days, inoculating 5 bacterial cakes to sterilized and cooled fungus No. 2 liquid culture medium (500 mL conical flask, 200 mL each flask) by using a puncher, and placing the culture strain in a constant temperature shaking table at 28 ℃ and 120rpm/min for 7 days to obtain trichoderma hook seed liquid. Inoculating the cultured seed solution to sterilized fungus No. 2 culture medium (maltose 20g, monosodium glutamate 10 g, glucose 10 g, yeast extract 3 g, corn steep liquor 1 g, mannitol 20g, KH 2 PO 4 0.5 g；MgSO 4 .7H 2 O 0.3 g；CaCO 3 20 g; 5-azacytidine 10 mg; distilled water 1L), inoculating 2.5. 2.5 mL seed solution per 100. 100 mL culture medium, and shake culturing the inoculated culture medium in a constant temperature shaker at 28deg.C and 120rpm/min for 14 days.
3. Extraction and isolation of Compounds of formula I
Centrifuging the cultured mycelium and fermentation broth with a high-speed centrifuge, soaking the fermentation broth in an equal volume of ethyl acetate for 24 hours, extracting for three times, collecting an ethyl acetate layer, concentrating under reduced pressure to obtain an ethyl acetate part extract, and obtaining 126.0g of total extract.
The extract is roughly separated by silica gel (200-300 meshes) column chromatography, petroleum ether-acetone (30:1, 20:1, 10:1,8:1,6:1,4:1,2:1, 1:1) is used as a mobile phase for elution, each gradient is 5 times of column volume, and the total of 8 components (Fr.1-Fr.8) are obtained by combining according to TLC results.
Fr.3 is eluted by an axial compression column (methanol: water-50-100%, gradient elution and 20 min) to obtain 6 components (Fr.3-1-Fr.3-6), fr.3-4 (retention time 13-15 min) is eluted by a gel column, pure methanol is used for elution, and the components are combined according to TLC detection results to obtain 5 components (Fr.3-4-1-Fr.3-4-5), and Fr.3-4-3 is purified by a semi-preparative liquid phase to obtain a compound shown as a formula I (the condition of the semi-preparative liquid phase purification is that Kromasil100-5-C18, 10X1250 mm, 4.5 mu m, akzo Nobel company, sweden): methanol (a): water (B); flow rate: 3 mL/min; detection wavelength: 190nm,210 nm, column temperature: 30 ℃;26% isocratic acetonitrile elution).
2. Identification of Compounds of formula I
The obtained compound is characterized by adopting methods of mass spectrum, infrared spectrum, nuclear magnetic resonance and the like, and as shown in figures 1-10, the specific results are as follows:
physical constants of compounds of formula I: ESI-MSm/z: 319.2268[M+H] + . The molecular weight of the compound was found to be 318, formula C 20 H 30 O 3 The unsaturation was 6.
Colorless oily form; c (C) 20 H 30 O 3 ；7.1 o (c 0.01, MeOH)；IR (KBr)v max 3433.1, 2936.7, 1735.6 cm -1 ；1H NMR (600 MHz, CDCl 3 ) δ: 4.40（1H, d,J= 18 Hz , H-20a）, 4.20（1H, d,J= 18 .2Hz, H-20b）, 3.98(1H, dd,J= 3.6, 6.6 Hz , H-3), 2.57（1H , d,J= 16.7 Hz, H-12a）, 2.46（1H , d,J= 16.9 Hz, H-12b）, 2.45（1H , m, H-5）, 2.42（1H , d,J= 16.9 Hz , H-4a）, 2.40（1H ,m, H-8a）, 2.14（1H , dd,J= 11.3, 8.9 Hz, H-14）, 2.00（1H , m, H-8b）, 1.97（1H , m,H-7a）, 1.90（1H , m, H-15a）, 1.84 (1H, dd,J= 8.2, 3.7 Hz, H-2), 1.51（3H, s, H-19）, 1.50（1H , d,J= 15.3 Hz, H-4a）, 1.32（3H, s, H-17）, 1.25（1H , m,H-7b）, 1.18（3H, d,J= 7.6 Hz, H-18）, 1.09（1H ,dd,J= 14.0, 9.3 Hz, H-15b）, 0.87（3H, s, H-16）； 13 C NMR (151 MHz, CDCl 3 ) δ: 200.2（C-11）, 154.0（C-9）, 148.9（C-10）, 74.4（C-3）, 67.3（C-20）, 58.7（C-12）, 51.4（C-14）, 50.4（C-5）, 49.6（C-2）, 45.8（C-）, 40.0（C-）, 34.4（C-4）, 30.4（C-7）, 28.2（C-5）, 27.6（C-15）, 26.8（C-16）, 24.6（C-8）, 23.6（C-17）, 21.6（C-19）, 21.4（C-18）；ESI-MSm/ z319.2268 [M+H] + (calculated value is C 20 H 31 O 3 + , 319.2268)。
As can be seen from the analysis of the characterization results, the structural formula of the isolated compound is as follows:
the technical scheme of the invention is further described through experiments.
Experimental example 1 antibacterial Activity of Compounds of formula I
1. Experimental method
3mg of the compound of formula I (prepared as in example 1) was weighed and dissolved in 0.1ml of DMSO to prepare 30000 ppm of mother liquor. A certain amount of mother solution is sucked by a pipetting gun, and 0.5 percent of Tween-80 sterile water is added according to the concentration to prepare the liquid medicine with the required concentration. 1mL of the liquid medicine is sucked by a pipette, placed in a sterilized culture dish, placed in 9mL of PDA again, shaken well and cooled. Taking out circular fungus cake with a puncher, picking up the circular fungus cake to the center of a culture dish with an inoculating needle, placing the culture dish in an incubator at 25 ℃ for culturing, and measuring the colony diameter after 48 hours.
Colony diameters were measured 3 days after the test treatment, and growth inhibition (%) was calculated.
D=D1－D2 （1）
Wherein: d represents colony growth diameter; d1 represents colony diameter; d2 represents the diameter of the bacterial cake.
Wherein: i represents the hypha growth inhibition rate; d0 represents the growth diameter of the control colony; dt represents the growth diameter of the drug-treated colonies.
2. Experimental results
The inhibition effect of the compound of the formula I on 8 common agricultural disease fungi is shown in the following table:
TABLE 1 inhibition of 8 common agricultural disease fungi by the compounds of formula I (%)
a. The concentration units are ppm.
The antibacterial experiments show that: the compound shown in the formula I has remarkable inhibitory activity on botrytis cinerea (the inhibition rate is 85.4%), sclerotium of rape (the inhibition rate is 79.6%), and has good inhibitory activity on strawberry anthrax (the inhibition rate is 68.2%) and corn stalk rot (the inhibition rate is 57.0%).
From the above examples and experimental examples, it can be seen that the present invention separates a novel compound from the secondary metabolite of trichoderma hook, which has a good inhibitory effect on various agricultural disease fungi. Thus, the compound and the extract containing the compound (e.g., fr.3-4-3, etc. of example 1) have application potential in inhibiting fungi of agricultural diseases and improving disease resistance of plants. The technical scheme of the invention is beneficial to the popularization and the efficient utilization of trichoderma hook and has good application prospect.
Claims (9)
1. A compound of formula I:
formula I.
2. A trichoderma hook fermentation broth extract, characterized in that: the trichoderma hook fermentation broth extract comprises the compound of claim 1.
3. The trichoderma hook fermentation broth extract according to claim 2, wherein: the trichoderma hook isTrichoderma hamatum。
4. Use of a compound according to claim 1, or a trichoderma hamatum fermentation broth extract according to claim 2 or 3, for inhibiting agricultural disease fungi.
5. Use according to claim 4, characterized in that: the agricultural disease fungi include at least one of the following fungi: gibberella Tritici, phytophthora capsici, sclerotinia sclerotiorum, botrytis cinerea, rhizoctonia solani, corn stalk base rot, strawberry anthrax, and peanut brown spot.
6. A pesticide for improving disease resistance of plants, characterized in that: which is prepared by using the compound according to claim 1 or the trichoderma hamatum fermentation broth extract according to claim 2 or 3 as an active ingredient.
7. A process for the preparation of a compound as claimed in claim 1, comprising the steps of:
step 1, extracting ethyl acetate part extract from fermentation broth of trichoderma hook;
step 2, coarsely separating the ethyl acetate part extract by column chromatography, wherein the volume ratio of the column chromatography is 30: 1-1: 1, petroleum ether-acetone is used as a mobile phase, 8 components are obtained through separation, and the components are sequentially recorded as Fr.1-Fr.8;
step 3, fr.3 is eluted by a radial compression column, and eluent is methanol aqueous solution with the volume fraction of 50-100% to obtain 6 components, which are sequentially recorded as Fr.3-1-Fr.3-6;
step 4, eluting Fr.3-4 by a gel column, wherein the eluent is methanol to obtain 5 components, which are sequentially recorded as Fr.3-4-1 to Fr.3-4-5;
and 5, taking Fr.3-4-3 for purification to obtain the final product.
8. The method of preparing as claimed in claim 7, wherein: the trichoderma hook isTrichoderma hamatum。
9. The method of preparing as claimed in claim 7, wherein: the preparation method of the ethyl acetate part extract comprises the following steps: separating fermentation broth of Trichoderma hook, soaking in ethyl acetate, extracting with ethyl acetate, mixing ethyl acetate phases, and concentrating under reduced pressure.
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